An Investigation Into the Culture Media Influence Within Plant Biotechnology.James Robert Hutchinson, Myerscough College.

Introduction: Plant hormones regulate many aspects of plant growth and development. Both auxin and cytokinin have been known for a long time to act either synergistically or antagonistically to control several significant developmental processes, such as the formation of meristem. A simplified version of these events is shown in figure three.Over the past few years, exciting progress has been made to reveal the molecular mechanisms underlying the auxin – cytokinin action and interaction (Su, et al., 2011). The framework for the complicated interaction of these two hormones in the control of leaf area and amount of shoot formation is the major focus of this poster.

Figure three: Overview of the Tissue Culture Process. It is drawn by the author courtesy of the Department of

Horticultural Sciences.

Graphs:

Results and Discussion: The number of leaves was the highest in treatment one (BAP 0.8mg/l¯1 and NAA 0.1mg/l¯1) which gave a mean average of 13.2. However, treatment one’s mean shoots were considerably lower (1.08) than treatments two and three where cytokinin and auxin were present. How does this work? The ratio of auxin to cytokinin plays an important role in the effect on plant growth. For instance, treatment four has no cytokinin or auxin involved and as such only has one shoot per plant whereas treatment three with 0.8mg/l¯1 of BAP and NAA respectively, has four! This observation supports a previous conclusion by Pernisova, et al. (2009) and their work involving these two phytohormones suggesting that auxin triggers organogenesis whereas cytokinin regulates it and/ or promotes plant cell division and growth. The results from the mean shoot per plant data indicate that cytokinin alone, or in small doses, has little effect on the parenchyma cells which promote shoot buds (Pernisova, et al., 2009). Auxin seems to regulate the biosynthesis of cytokinin and the two act in concert with one another. This can be seen more clearly in Plate one where there are more shoots in group three’s plant (four shoots) as opposed to group four’s (no hormones added) one shoot. Based on the data, it is suggested that BAP 0.8mg/l¯1 and NAA 0.8mg/l¯1 would be the best way to harvest Cotinus after five weeks in culture and thus possibly used as a source to attempt several in vitro ways to breed this attractive plant.

Materials and Methodology: This research was carried out at the tissue culture laboratory of Myerscough College, England, over a five week period during the Winter of 2011.

Figure one: Effects of combinations of different levels of cytokinin (BAP) and auxin (NAA) on the number of leaves in vitro propagation of

Cotinus coggygyria. Bars represent standard error.

Figure two: Interaction of cytokinin (BAP) and auxin (NAA) during somatic embryogenesis of Cotinus coggygyria shoots.

Plate one: Selected Cotinus after four weeks growth.

References: 1. Murashige, T. Skoog, F. (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia planatarium 15: 473 – 479.

2. Pernisova, M. Klima, P. Horak, J. Valkova, M. Malbeck, J. Soucek, P. Reichman, P. Hoyerova, K. Dubova, J. Frimi, J. Zimalova, E. Hejatko, J., 2008. Cytokinins modulate auxin – induced organogenesis in plants via regulation of the auxin efflux. Proceedings of the National Academy of Sciences of the USA, [online].Available at: http://www.pnas.org/content/106/9/3609.full [accessed 25 February 2012]. 3. Su,Y.H. Liu, B.U. Zhang, X.S., 2011. Auxin – Cytokinin Interaction Regulates Meristem Development. Molecular Plant., [online]. Available at: http://mplant.oxfordjournals.org/content/early/2011/02/28/mp.ssr007.full [accessed 17 February 2012].

Week One

• The Cotinus plantlets were obtained from the culture room of the college. The nutrient media contained minerals, macro and micro salts according to Murashige and Skoog (1962) with sucrose added at 30g/l. In addition, BAP and NAA were added to the MS medium in different concentrations (including a control group). The pH of the medium was adjusted to 5.8. It

was then dispensed into culture vessels and autoclaved at 15psi for 15 minutes, allowed to cool then poured into containers; 12 – 15ml into the

small plastic vials and about 50ml into the large tub containers.

Week Two

• The pre prepared explant culture media was checked for contamination and each pot was recorded for weight. The sub – cultured Cotinus tissue was

cut into relevant sized pieces, transferred to pots and the weight recorded. The pots were incubated at 25°c ± 1°c in a growth cabinet and observed at

weekly intervals.

Week Five

• *Readings were taken after four weeks incubation for: • Number of plantlets sub – cultured into each tub• Dry weight of shoots• Dry weight of roots/ callus• Number of shoots per plant• Mean dry weight per shoot• Leaf area

*Note: For ease of reading, two graphs are shown to represent the findings of the experiment; mean average leaf area data and mean shoots per plant..